fermentation Article Microbial Composition of Fermented Korean Soy Paste (Doenjang) Prepared by Adding Different Herbs during Fermentation Mohamed Mannaa 1,2, Seong-Soon Cho 3, Young-Su Seo 1,* and Inmyoung Park 3,* 1 Department of Integrated Biological Science, Pusan National University, Busan 46241, Korea; [email protected] 2 Department of Plant Pathology, Cairo University, Giza 12613, Egypt 3 School of Food and Culinary Arts, Youngsan University, Busan 48015, Korea; [email protected] * Correspondence: [email protected] (Y.-S.S.); [email protected] (I.P.); Tel.: +82-51-510-2267 (Y.-S.S.); +82-51-540-7236 (I.P.) Abstract: The microbiota involved in Korean soy paste (doenjang) fermentation are the key factors determining its quality aspects. In this study, doenjang was prepared by adding three different herbs (Peppermint, Korean mint, and Coriander), and their effect on the microbiota composition was evaluated by 16S rRNA metagenomic analyses. The β-diversity statistics indicated clear distinctions in the doenjang microbiota after the addition of herbs. A microbial composition analysis revealed that Tetragenococcus was among the dominant genera in the four doenjang groups, with a relatively higher abundance in the Korean mint group. In the Peppermint and Korean mint doenjang groups, the levels of undesirable microbes, such as opportunistic pathogens belonging to the genera Sphingobacterium and Pantoea, were significantly reduced. Additionally, other desirable microbes that are known Citation: Mannaa, M.; Cho, S.-S.; Seo, to exhibit beneficial properties and produce bioactive compounds, such as Saccharopolyspora and Y.-S.; Park, I. Microbial Composition Buttiauxella, were present at significantly higher levels. Significant negative correlations between of Fermented Korean Soy Paste members of the Bacillaceae and Halomonadaceae, Lactobacillaceae and Tissierellaceae, and the La- (Doenjang) Prepared by Adding cobacillaceae and Erwiniaceae families were observed, indicating possible antagonistic relationships. Different Herbs during Fermentation. Taken together, our results demonstrated that the incorporation of herbs, particularly Peppermint and Fermentation 2021, 7, 93. https:// Korean mint, during doenjang fermentation resulted in significant shifts in the microbial composition doi.org/10.3390/fermentation7020093 and could be utilized for beneficial effect on its fermentation. Academic Editors: Annalisa Serio and Clemencia Chaves-López Keywords: fermented soy paste; metagenomics analysis; coriander; peppermint; Korean mint Received: 14 May 2021 Accepted: 8 June 2021 Published: 10 June 2021 1. Introduction Fermentation has been practiced for thousands of years for the preservation and Publisher’s Note: MDPI stays neutral storage of food materials [1]. Fermented foods are important sources of nutrition and with regard to jurisdictional claims in have improved flavors; they are known for their potential health promoting and disease published maps and institutional affil- preventing properties, which contribute to their current worldwide popularity [2]. iations. Doenjang is a traditional Korean fermented soy paste that has been consumed for thousands of years as a protein source and a basic seasoning ingredient. Traditionally, doenjang production is a two-stage process that is initiated by preparing meju, a dry fermented, moldy cooked soybean block, using naturally occurring bacteria and fungi. Copyright: © 2021 by the authors. The second stage is performed by soaking the meju in salty water in porcelain containers Licensee MDPI, Basel, Switzerland. under sunlight for several weeks to produce a solid portion that is called soybean paste This article is an open access article (doenjang) and a liquid portion called soy sauce (gangjang). The doenjang is then mashed distributed under the terms and and further fermented before consumption through a few weeks to several years [3]. conditions of the Creative Commons Doenjang has attracted much attention and popularity due to its health-related ben- Attribution (CC BY) license (https:// eficial properties such as antioxidative, anti-inflammatory, immunity enhancing, and creativecommons.org/licenses/by/ fibrinolytic activities [4–6]. Moreover, doenjang consumption may be associated with a 4.0/). Fermentation 2021, 7, 93. https://doi.org/10.3390/fermentation7020093 https://www.mdpi.com/journal/fermentation Fermentation 2021, 7, 93 2 of 12 lower incidence of type 2 diabetes, greater antimutagenic and anticancer activity, and an antiobesogenic effect [7–10]. The properties of doenjang including the health promotion effects and unique flavors and taste can be attributed to several bioactive compounds (e.g., bioactive peptides, isoflavones, saponins, phytic acid, and linoleic acid), produced as the result of the degradation and decomposition of soybean proteins and additional ingredients by the fermentation microbes [5,11,12]. Thereby, the microbes involved in this fermentation are the most critical components for understanding the phenomenon associated with the quality characteristics and safety aspects of doenjang [13,14]. The microbial composition of doenjang can be affected by several factors, mainly the environmental variables and the basic ingredients during fermentation [15]. Culture-dependent and culture-independent microbiological studies have attempted to reveal the microbial community structure in doenjang during fermentation. The culture-based methods indicated that members of the Bacillus genus, such as B. subtilis, B. licheniformis, and B. megaterium, were among the dominant bacteria, and members of the Aspergillus, Mucor, and Rhizopus genera were among the dominant fungi in doenjang and were considered to play important roles during fermentation [11,16,17]. The bacterial community of doenjang underwent a shift toward salt-tolerant bacteria as the fermenta- tion of doenjang progressed, during which Bacillus spp., the lactic acid bacterium (LAB) Tetragenococcus halophilus, and coagulase-negative staphylococci were predominant [18]. However, these studies used culture-based methods and were limited in the detection and identification of the involved microbiota. Culture-independent techniques such as 16S rRNA libraries and denaturing gradient gel electrophoresis (DGGE) have been used to study doenjang microbiota, and unlike in the case of previous studies, other microbes such as Staphylococcus equorum, Leuconostoc mesenteroides, T. halophilus, and Enterococcus faecium were found to be dominant [19,20]. More recently, an rRNA pyrosequencing study revealed that Bacillus spp., among bacteria, and Mucor spp., among fungi, were predominant in doenjang during the early fermentation period. However, as fermentation progressed, changes were observed in the microbial composition and bacteria such as Ignatzschineria, Myroides, Enterococcus, Corynebacterium, and Clostridium spp., and fungi such as Geotrichum, Scopulariopsis, Monascus, Fusarium, and Aspergillus spp. increased in abundance [21]. Another recent study showed that the phylum, Firmicutes, was the dominant taxon in both the traditional and modified doenjang samples; LAB, including mainly Tetragenococcus, Enterococcus, Leuconostoc, and Lactobacillus spp., were detected in most samples, whereas among the non-LAB, Staphylococcus and Ochrobactruma spp. were dominant [22]. In the same line, a recent study utilized an Illumina MiSeq platform to study the microbial composition of doenjang and revealed that members of the halophilic or halotolerant Tetragenococcus, Staphylococcus, Debaryomyces, Meyerozyma, Millerozyma, and Hyphopichia were predominant [23]. Herbs and spices have had a long history of use in foods as natural additives for improving health and sensory properties and are becoming more attractive as potential alternatives to pharmaceutical agents that provide significant protection against chronic diseases [24]. Various types of herbs such as Peppermint (Mentha piperita), Korean mint (Agastache rugosa), and Coriander (Coriandrum sativum) have been studied and are shown to be sources of a range of bioactive phytochemicals with a multitude of properties, including antimicrobial, antioxidant, and anticancer activities [25–27]. Hence, it is of great importance to investigate the role of different herbs and to demonstrate the positive effects of their incorporation in food. A study on the microbes involved in doenjang fermentation and the effect of the ingredients used is crucial to understanding the fermentation process and the quality of the produced doenjang. Therefore, the objective of this study was to use high-throughput next generation sequencing-based metagenomics analyses to evaluate the effects of adding different herbs (Peppermint, Korean mint, and Coriander) on the microbial composition of doenjang. Controlling the process of doenjang fermentation by supplementation with dif- Fermentation 2021, 7, 93 3 of 12 ferent ingredients may result in the establishment of customized amounts and compositions of ingredients with respect to the effects on the microbes involved in the fermentation. 2. Materials and Methods 2.1. Preparation of Fermented Soy Paste Samples Fermented doenjang was prepared using the traditional Korean method under the guidance of a master craftsman of Korean cuisine at Youngsan University located in Pusan, Korea. Briefly, fermented doenjang-meju bricks (approximately 2 kg, Gigang County, Korea) were soaked in 6 L of a 20% (w/v) solar salt (Shinan, Korea) solution in a porcelain pot, and then 3 pieces of pure charcoal